Pharmacokinetics of the Couple of Radix Aconiti Lateralis Preparata and Cinnamomum Cassia in Rats with Osteoporosis with Kidney-yang Deciency by UPLC and Microdialysis Method

Background: To conrm the metabolites of the Aconite and Cinnamon herb couple in rat, and characterize its pharmacokinetics. Methods: Microdialysis probes were inserted into the jugular vein and knee joint of Sprague Dawley rat, and dialysates of different administration groups were collected. Target analytes were separated on a hydrophilic interaction liquid chromatography column (ACQUITY UPLC BEH C8 2.1×100 mm, 1.7 μm) and analyzed with an ultra-performance liquid chromatography (UPLC) under multiple reaction monitoring modes. Results: The experiment shows that the concentrations of quality of the three metabolites in mixing extraction of the herb couple were 0.3701% for trans-cinnamic acid, 0.1249 % for mesaconitine, and 0.0469 % for hypoaconitine, and Cinnamon group was 0.1731 % for trans-cinnamic acid, Aconite group were 0.0017 % for mesaconitine, and 0.0300 % for hypoaconitine. The concentrations of quality of the metabolites in rat plasma of the herb couple group were 0.0028% for trans-cinnamic acid, 0.0947% for mesaconitine, and 0.1124% for hypoaconitine. And the concentrations of quality of Aconite group and Cinnamon group were 0.0019% for trans-cinnamic acid, 0.0307% for mesaconitine, and 0.0220% for hypoaconitine. Pharmacokinetic results showed that the mean half-lives of the microdialysis samples of blood of Cinnamon group was 492.18 min for trans-cinnamic acid, and Aconite group were 102.48 min for mesaconitine and 93.27 min for hypoaconitine, and the herb couple ware 181.36 min for trans-cinnamic acid, 103.9 min for mesaconitine and 116.01 min for hypoaconitine, and the microdialysis samples of joint of Cinnamon group was 190.85 min for trans-cinnamic acid, and Aconite group were 48.51 min for mesaconitine and 46.01 min for hypoaconitine, and the herb couple was 131.19 min for trans-cinnamic acid, 49.36 min for mesaconitine and 146.68 min for hypoaconitine. Conclusions: The mixed extraction of aconite and cinnamon can promote the dissolution of the active ingredients. The herb couple can promote the absorption of the active ingredients, improve the distribution of the active ingredients in the joint and blood, prolong the half-lives of the active ingredients. It shows that the compatibility of aconite and cinnamon can increase the bioavailability of the drug and improve the clinical ecacy.


Introduction
Osteoporosis (OP) is a systemic metabolic bone disease characterized by bone loss and degeneration of bone microstructure [−] . The traditional theory of traditional Chinese medicine holds that OP is closely related to the de ciency of kidney [−] . Studies have shown that kidney-yang de ciency is an important factor in the occurrence of OP [] . Kidney de ciency can cause hypothalamic-pituitary-gonadal axis hypofunction, decreased sex hormone secretion, hyperabsorption of bone, decreased secretion of calcitonin, and decreased intestinal calcium absorption, resulting in the decrease of osteogenesis and bone tissue mass. The treatment of primary OP is mainly based on western medical methods. These methods have good prevention and treatment effects, but some serious adverse reactions can't be avoided, limiting their clinical applications [−] . Traditional Chinese medicine has fewer side effects, which has been used to treat kidney-yang de ciency. Traditional Chinese medicine therapy is a very promising for OP treatment [] . However, due to the complex mechanism, the development of traditional Chinese medicine therapy is restricted. Therefore, the mechanism of traditional Chinese medicine t needs to be further studied [−] .
Aconite and Cinnamon herb were rst recorded in "Shennong Herbal Classic" [−] . Aconite is the processed product of Aconitum carmichaelii Debeaux tuber. The components of Aconiti are mainly diester alkaloids, including aconitine, mesaconitine and hypoaconitine, etc., which have analgesic and anti-in ammatory effects [−] . Stadies show that it can in uence energy metabolism and gene expression spectrum in rats and activate the proliferation of bone marrow mesenchymal stem cells of mouse [−] . Cinnamon is the dried bark of Cinnamomum cassia Presl [−] . The main content of Cinnamon is trans-cinnamic acid [] . According to pharmacological research, trans-cinnamic acid has antibacterial and anti-in ammatory effects [−] . Studies have found that trans-cinnamic acid has in uence on bone marrow mesenchymal stem cell proliferation and osteoblast differentiation of rat [−] . The compatibility of Aconite and Cinnamon can improve kidney de ciency and spleen de ciency [] , and the hormone level of hypothalamus-pituitary-adrenal cortex axis (HPA) in rats with kidney yang de ciency [−] .
The herb couple was rst recorded in Treatise on Febrile and Miscellaneous Diseases [−] . Relevant studies have shown that both Aconite and Cinnamon can be used to treat OP separately, but the mechanism of the herb couple in the treatment of OP with kidney-yang de ciency need to be revealed [] .
In this paper, the PK-PD model and microdialysis sampling coupled with UPLC were used to study the active ingredients of the aconite, cinnamon, and the compatibility of the two. The metabolites of the herb couple were con rmed, its pharmacokinetics was characterized. This work may provide a basis for the clinical application of aconite and cinnamon herb couple to treat OP of kidney-yang de ciency syndrome.

Chemicals
All reagents were of analytical grade unless speci ed otherwise. Water (18.2 MΩ cm) was produced by a laboratory Milli-Q pure water meter.

Animals
Sprague Dawley (SD) rats were purchased from the Animal Experimental Center of Zhejiang Chinese Medicine University. They were raised in the Animal Experimental Center of Zhejiang Chinese Medicine University (license number: 20180006004067). Before experimentation, the rats were allowed a 1-week acclimation period in the animal house using the independently isolated feeding cages with natural illumination. The rats were fed standard food and puri ed water. The environment temperature was kept at 25 ± 2°C, and the humidity was ≥ 40 %.
2.3.2 Establishment of pathological model of OP with kidney-yang de ciency SD rats were randomly grouped according to their body weight. Anesthetized with ip10% chloral hydrate 3mL/kg, then x the animal on the operating table. Through bilateral ophorectomy, the fallopian tubes and accompanying blood vessels are ligated rst, and then the "mulberry-like" ovaries are separated with elbow surgical clamps, ligated, and cut off. The rats were injected with 40,000 U/mL penicillin every day after the operation, each injection was 1 mL/d, and the injection was continued for 3 days, and then the rats were routinely reared. Starting from the second week, the experimental group was injected with hydrocortisone injection (5 mg/mL) 20 mg/kg on the hind limbs every day. From the 4th week to the 8th week, the rats in the experimental group were injected with hydrocortisone injection twice a week to maintain the state of kidney-yang de ciency. Generally based on rat behavior indicators, serum E2, T, ACTH, cAMP, cGMP, etc. levels, bone density and bone mineral content and other tests. Judge whether the model of OP with kidneyyang de ciency type is successful. The rats were divided into Aconite group, Cinnamon group and the herb couple (1:1). The rats were a successful model and anesthetized. Rat body temperature was maintained at 37°C using a heating pad during the experiment, and their heads were xed on the brain stereotaxic locator to x the limbs of the rats. In the rst step, to make an incision at the upper left of the left clavicle, then to nd the jugular vein, insert the torn tube into the vein, then place the blood probe in the venous blood vessel, and suture the skin wound carefully. In the second step, insert the traction needle into the cartilage of the rat's leg joint cavity, and insert the probe into the overlap of the traction needle and articular cartilage carefully, so that the probe membrane of the joint probe is partially placed subcutaneously.

microdialysis experiment
To use Ringer's solution to prime and balance 30 ~ 60 min. Aconite and Cinnamon 1 mL / 100g were injected into the duodenum according to the weight of each rat. The samples of the jugular vein and joint dialysate were collected by cryopreservation sampler after administration at different times at 10 min 20nin 30min 40min 50min 60min 120min 180min 240min 300min 360min 480min, a total of 8 h.
After all the samples are collected, the last dose, half an hour after administration, to take out rat whole blood, to wait for it to stand and layer, to separate the supernatant and store it in -20℃ refrigerator.

Preparation of standard and unmeasured solutions and Determination
Preparation of standard solutions Precision weighing standard mesaconitine 4.60 mg, hypoaconitine 4.70 mg, and trans-cinnamic acid standard 2.26 mg, with Chromatography Acetonitrile and methanol at 10 mL capacity bottle, to dilute ten times and store at 4°C.

Preparation of blood samples group
To precision absorption the blood 60 uL of different components, precision addition 20 uL ammonia water, vortex 2 min, precision addition 520 uL Chromatography Acetonitrile and methanol to precipitation protein, and vortex 3 min, 15000 r/min centrifuges for 10 minutes. Take supernatant, use 0.22 um organic lter membrane to lter.
And to analyze them according to the chromatographic conditions respectively, and record the peak area data. Calculate the content value of each group.

Method validation and Determination
There is the linearity of the standard solution, preparation of reference standard solution method was 0.2 mL 0.4 mL 0.6 mL 0.8 mL 1 mL 2 mL 4 mL 5 mL in 5 mL capacity bottle, Volume to scale with Chromatographic methanol and Chromatography Acetonitrile (1:1) solution, different concentrations of mixed reference solution were obtained and ltered into the injection bottle with a disposable syringe to use 0.22 um organic lter membrane to lter. According to chromatographic conditions to inject the sample, determine the peak area. With the peak area as the ordinate and the concentration of each group of samples as the abscissa to draw a standard curve. The speci city of the method was determined by comparing the chromatograms of blanks with those corresponding to the samples. The precision of the method was determined by taking each group of blood samples, according to the chromatographic conditions for 8 times, and to determine the peak area of blood samples. To take the same batch of the test solution and analyze it at 0, 2, 4, 6, 8, 12, 16, 18, 24 h according to the chromatographic conditions to determine the stability of the method, and record the peak area data. The repeatability of the method was determined by preparing the same batch of blood for 8 groups, then the peak area of the peak plasma sample was determined according to the chromatographic conditions.

Freeze-dried powder production and determination
Preparation of freeze-dried powder of Aconiti or Cinnamon: To use 240g Aconiti or 80g Cinnamon with water as a solvent to condense and re ux for extraction twice respectively. Put it in a freeze dryer to freezedrying, quickly weigh and transfer the powder to a plastic bag, and store at 4℃ in the refrigerator.
To weigh freeze-dried powder of Aconiti 1.0085g, freeze-dried powder of Cinnamon 0.5081g and 0.1096g freeze-dried powder of the herb couple precisely, To draw 20 ul formic acid to add, and x to 20 mL volumetric ask with methanol, to vortex for 5min, after standing for 1h, to ultrasound for 30min, and to centrifugation (10000r/min, 10min) to separate the supernatant, use 0.22 um organic lter membrane to lter. And analyze them according to the chromatographic conditions respectively, and record the peak area data.

Statistical analysis
All statistical analyses were performed using the SPSS software (Version 13.0). P-values less than 0.05 were considered statistically signi cant.  Selectivity: No interferences from endogenous substances were observed at the retention times of each respective analyte (Fig. 1), trans-cinnamic acid, mesaconitine and hypoaconitine were eluted at 7.5, 11.9 and 15.3 min, respectively.
Precision: The calculated RSD values of the blood samples in each group are 0.4674%, 1.3080% and 1.7328%, and the RSD values are less than 2%, which indicates that the precision of the instrument method is good.
Stability: The calculated RSD values of the peak areas of trans-cinnamic acid, mesaconitine and hypoaconitine are 1.5489%, 1.9260% and 1.9556%, respectively. And their RSD value is less than 2%, the data showed that the three components in the sample solution were stable within 24 hours after preparation.
Repeatability: The RSD values of each component peak area of trans-cinnamic acid, mesaconitine and hypoaconitine were calculated as follows: 1.8659%, 1.4124% and 1.2253%, respectively. And their RSD value is less than 2%, which indicates that the repeatability of the method is good. In short,the precision and accuracy data in Table 1 indicated that the present UPLC method showed good accuracies and reproducibilities.

powder rate of Frozen Dry Powder
Calculate the powder rate separately: [the weight of the dry powder after drying (including the weight of the evaporating dish)-the weight of the evaporating dish (after drying)] / the quality of the pieces. Available as shown in Table 2.

Determination of Frozen Dry Powder
The peak area external standard method combined with the powder extraction rate was used to calculate the content of each component in each group of freeze-dried powder medicinal materials, as shown in

Determination of blood content in each group
The peak area external standard method was used to calculate the content of each component in the blood of each group, as shown in Fig. 3. Under the premise that the combination of the herb couple, the content of trans-cinnamic acid, mesaconitine and hypoaconitine of the herb couple were signi cantly higher than trans-cinnamic acid of Aconite group and mesaconitine and hypoaconitine of Cinnamon group in the early stage, the combined administration of the herb couple was tested after absorption in the body, and it was concluded that trans-cinnamic acid, mesaconitine and hypoaconitine of the herb couple were higher than trans-cinnamic acid of Aconite group and mesaconitine and hypoaconitine of Cinnamon group (P < 0.001). Drug metabolism contributes substantially to interindividual differences in drug response and is also often involved in drug interactions, resulting in either therapeutic failure or adverse effects [] .
Pharmaceutical interactions are caused by the chemical, physical or physicochemical incompatibility of drugs or adjuvants used. These can even occur outside the body and during concomitant administration via the same route [] . It can be seen that the combination of the herb couple may exert a synergistic effect, which can promote the absorption of active ingredients, further increase the bioavailability of the drug and thus improve the clinical e cacy, which makes the application of Chinese medicine compatibility particularly important.

Determination of the microdialysis samples of blood and joint
The peak area external standard method was used to calculate the content of the components of the microdialysis samples at different time points, and then the average content at each time point was calculated. To take the time of the microdialysis samples connected as the abscissa, and the content of the microdialysis samples at each time point as the ordinate, as shown in Fig. 4 and Fig. 5, and the relevant kinetic parameters are shown in Table 3. Trans-cinnamic acid, mesaconitine and hypoaconitine were rapidly accessed to plasma and Joint cavity, and the concentration peaked of the microdialysis samples of blood of Cinnamon group was 60 min for trans-cinnamic acid, and Aconite group were 120 min for mesaconitine and 120 min for hypoaconitine, and the herb couple ware 30 min for trans-cinnamic acid, 60 min for mesaconitine and 60 min for hypoaconitine. And the concentration peaked of the microdialysis samples of joint of Cinnamon group was 50 min for trans-cinnamic acid, and Aconite group were 120 min for mesaconitine and 120 min for hypoaconitine, and the herb couple was 30 min for trans-cinnamic acid, 60 min for mesaconitine and 60 min for hypoaconitine, respectively. In the microdialysis samples of blood and joints, the action time of trans-cinnamic of the herb couple was 20 minutes earlier than Cinnamon group, and the action time of mesaconitine and hypoaconitinethe of the herb couple were 10 minutes earlier than Aconite group (P < 0.05). In the microdialysis samples of blood and joints, compared with Cinnamon group, the action time of trans-cinnamic of the herb couple can hold on for about 80 min longer (P < 0.05). And compared with Aconite group, the action time of mesaconitine and hypoaconitinethe of the herb couple can hold on for about 130 min longer (P < 0.05). The pharmacokinetic study of monomeric trans-cinnamic acid reported that the peak time of trans-cinnamic acid by gavage was about 30 min, and the t1/2 was about 36 min [] .
After oral administration of aconitine to SD rats, the blood concentration in the body is eliminated quickly, and individual differences are large. The t1/2 after the percutaneous replacement is about 360 min, and the peak time after oral administration is 15 min [−] . To observe the difference in the content of the microdialysis samples at different times and under different administration conditions. It can be seen that the contents of trans-cinnamic acid of the herb couple was signi cantly higher than Cinnamon group, and mesaconitine and hypoaconitine of the herb couple were signi cantly higher than Aconite group in the microdialysis samples of blood and joint cavity. From the peak content of trans-cinnamic, mesaconitine and hypoaconitinethe, the content of the synovial uid is slightly more than that in the blood as shown in Fig. 4 and Fig. 5. Among them, the highest is that the hypoaconitine of cinnamon group in the microdialysis samples of joints is 0.303% more than that of cinnamon group in the microdialysis samples of blood, and the lowest is that the trans-cinnamic of cinnamon group in the microdialysis samples of joints is 0.01% more than that of cinnamon group in the microdialysis samples of blood. The effective time of trans-cinnamic acid of the herb couple was 150 min, and that of Cinnamon group was 230.
Mesaconitine and hypoaconitine of the herb couple were 360 min, and that of Aconite group was 200 min. The effective time of trans-cinnamic acid of the herb couple was signi cantly longer than Cinnamon group, and mesaconitine and hypoaconitine of the herb couple were signi cantly higher than Aconite group (P < 0.05). It can be seem that the herb couple can improve the distribution of the active ingredients in the joint and blood, the absorption of the active ingredients in the body is improved, and the time for the effective substances to be distributed in the body is prolonged, making the drug action more durable (P < 0.05). According to the above, the mixed extraction of aconite and cinnamon can promote the dissolution of the active ingredients. It can provides great help for the subsequent absorption and utilization of the drug in the body. Combined with the change in the content of the drug in the body, it shown that the compatibility of Aconite and Cinnamon may play a synergistic effect, promote dissolution and enhance absorption, further improve the bioavailability of Aconite and Cinnamon and improve the clinical e cacy, which makes the application of Aconite and Cinnamon compatibility particularly important.

Conclusion
The mixed extraction of aconite and cinnamon can promote the dissolution of the active ingredients. The herb couple can promote the absorption of the active ingredients, improve the distribution of the active ingredients in the joint and blood, prolong the half-lives of the active ingredients. It shows that the compatibility of aconite and cinnamon can increase the bioavailability of the drug and improve the clinical e cacy. Content of freeze-dried powder in each group A. trans-cinnamic acid B. mesaconitine C. hypoaconitine Compared to the single herb groups, ***p<0.001.

Figure 3
Contents of blood components in each group A. trans-cinnamic acid B. mesaconitine C. hypoaconitine Compared to the single herb groups, ***p<0.001.

Figure 4
Changes in the contents of the microdialysis samples of blood in different groups